Human oral mucosa-derived neural crest-like stem cells differentiate into functional osteoprogenitors that contribute to regeneration of critical size calvaria defects.

BACKGROUND AND OBJECTIVE: Regeneration of large bony defects is an unmet medical need. The therapeutic effect of fully developed bony constructs engineered in vitro from mineralized scaffold and adult stem cells is hampered by deficient long-term graft integration. The purpose of the present study was to investigate the regenerative capacity of a bony primordial construct consisting of human oral mucosa stem cells (hOMSC)-derived osteoprogenitors and absorbable Gelfoam® sponges. METHODS: Gingiva and alveolar mucosa-derived hOMSC were differentiated into osteoprogenitors (Runx2 and osterix positive) and loaded into Gelfoam® sponges to generate primordial hOMSC constructs. These were implanted into critical size calvaria defects in the rat. Defects treated with human dermal fibroblasts (HDF) constructs; Gelfoam® sponges and untreated defects served as controls. RESULTS: After 120-day post-implantation defects treated with hOMSC constructs, HDF constructs and gelatin and untreated defects exhibited 86%, 30%, 21%, and 9% of new bone formation, respectively. Immunofluorescence analysis for human nuclear antigen (HNA), bone sialoprotein (BSP), and osteocalcin (OCN) revealed viable hOMSC-derived osteoblasts and osteocytes that formed most of the cell population of the newly formed bone at 30 and 120 days post surgery. Few HNA-positive HDF that were negative for BSP and OCN were identified together with inflammatory cells in the soft tissue adjacent to new bone formation only at 30 days post implantation. CONCLUSION: Collectively, the results demonstrate that primordial in vitro engineered constructs consisting of hOMSC-derived osteoprogenitors and absorbable gelatin almost completely regenerate critical size defects in an immunocompetent xenogeneic animal by differentiating into functional osteoblasts that retain the immunomodulatory ability of naïve hOMSC.

Human Oral Mucosal Stem Cells Reduce Anastomotic Leak in an Animal Model of Colonic Surgery.

BACKGROUND: Anastomotic leak is regarded as one of the most feared complications of bowel surgery; avoiding leaks is a major priority. Attempts to reduce or eliminate leaks have included alternate anastomotic techniques. Human oral mucosa stem cells (hOMSC) are self-renewing and expandable cells derived from buccal mucosa. Studies have shown that hOMSC can accelerate tissue regeneration and wound healing. The objective of this study was to evaluate whether hOMSC can decrease anastomotic leak rates in a murine model of colon surgery. METHODS: Two experiments were performed. In the first study, mice underwent colonic anastomosis using five interrupted sutures. hOMSC (n = 7) or normal saline (NS; n = 17) was injected into the colon wall at the site of the anastomosis. To evaluate whether hOMSC can impact anastomotic healing, the model was stressed by repeating the first experiment, reducing the number of sutures used for the construction of the anastomosis from five to four. Either hOMSC (n = 8) or NS (n = 20) was injected at the anastomosis. All mice that survived were sacrificed on postoperative day 7. Anastomotic leak rate, mortality, daily weight, and daily wellness scores were compared. RESULTS: In the five-suture anastomosis, there were no differences in anastomotic leak rate, mortality, or daily weight. Mice that received hOMSC had significantly higher wellness scores on postoperative day 2 (p < 0.05). In the four-suture anastomosis, there was a significant decrease in leak rate (70% [NS] vs. 25% [hOMSC], p = 0.029) and higher wellness scores in mice that received hOMSC (p < 0.05). CONCLUSION: Our study suggests that injecting hOMSC at the colonic anastomosis can potentially reduce anastomotic leak and improve postoperative wellness in a murine model of colon surgery.

Examination of the Therapeutic Potential of Mouse Oral Mucosa Stem Cells in a Wound-Healing Diabetic Mice Model.

Diabetic wounds' delayed healing response is still considered a major therapeutic challenge. Stem cells and derived cellular products have been an active field of research for novel therapies referred to as regenerative medicine. It has recently been shown that human oral mucosa stem cells (hOMSCs) are a readily accessible source for obtaining large quantities of stem cells. This study evaluates the potential of mouse oral mucosa stem cells (mOMSCs) to enhance wound healing in a diabetic (db/db) mouse model by morphological and histological analysis. We show that mOMSCs-treated wounds displayed a significantly faster wound-healing response (p ≤ 0.0001), featuring faster re-epithelialization and a larger area of granulation tissue (p ≤ 0.05). Taken together, these results suggest that oral mucosa stem cells might have therapeutic potential in diabetic wound healing.

Implantation of 3D Constructs Embedded with Oral Mucosa-Derived Cells Induces Functional Recovery in Rats with Complete Spinal Cord Transection.

Spinal cord injury (SCI), involving damaged axons and glial scar tissue, often culminates in irreversible impairments. Achieving substantial recovery following complete spinal cord transection remains an unmet challenge. Here, we report of implantation of an engineered 3D construct embedded with human oral mucosa stem cells (hOMSC) induced to secrete neuroprotective, immunomodulatory, and axonal elongation-associated factors, in a complete spinal cord transection rat model. Rats implanted with induced tissue engineering constructs regained fine motor control, coordination and walking pattern in sharp contrast to the untreated group that remained paralyzed (42 vs. 0%). Immunofluorescence, CLARITY, MRI, and electrophysiological assessments demonstrated a reconnection bridging the injured area, as well as presence of increased number of myelinated axons, neural precursors, and reduced glial scar tissue in recovered animals treated with the induced cell-embedded constructs. Finally, this construct is made of bio-compatible, clinically approved materials and utilizes a safe and easily extractable cell population. The results warrant further research with regards to the effectiveness of this treatment in addressing spinal cord injury.

Dopaminergic-like neurons derived from oral mucosa stem cells by developmental cues improve symptoms in the hemi-parkinsonian rat model.

Achieving safe and readily accessible sources for cell replacement therapy in Parkinson's disease (PD) is still a challenging unresolved issue. Recently, a primitive neural crest stem cell population (hOMSC) was isolated from the adult human oral mucosa and characterized in vitro and in vivo. In this study we assessed hOMSC ability to differentiate into dopamine-secreting cells with a neuronal-dopaminergic phenotype in vitro in response to dopaminergic developmental cues and tested their therapeutic potential in the hemi-Parkinsonian rat model. We found that hOMSC express constitutively a repertoire of neuronal and dopaminergic markers and pivotal transcription factors. Soluble developmental factors induced a reproducible neuronal-like morphology in the majority of hOMSC, downregulated stem cells markers, upregulated the expression of the neuronal and dopaminergic markers that resulted in dopamine release capabilities. Transplantation of these dopaminergic-induced hOMSC into the striatum of hemi-Parkinsonian rats improved their behavioral deficits as determined by amphetamine-induced rotational behavior, motor asymmetry and motor coordination tests. Human TH expressing cells and increased levels of dopamine in the transplanted hemispheres were observed 10 weeks after transplantation. These results demonstrate for the first time that soluble factors involved in the development of DA neurons, induced a DA phenotype in hOMSC in vitro that significantly improved the motor function of hemiparkinsonian rats. Based on their neural-related origin, their niche accessibility by minimal-invasive procedures and their propensity for DA differentiation, hOMSC emerge as an attractive tool for autologous cell replacement therapy in PD.

Astrocyte-like cells derived from human oral mucosa stem cells provide neuroprotection in vitro and in vivo.

Human oral mucosa stem cells (hOMSC) are a recently described neural crest-derived stem cell population. Therapeutic quantities of potent hOMSC can be generated from small biopsies obtained by minimally invasive procedures. Our objective was to evaluate the potential of hOMSC to differentiate into astrocyte-like cells and provide peripheral neuroprotection. We induced hOMSC differentiation into cells showing an astrocyte-like morphology that expressed characteristic astrocyte markers as glial fibrillary acidic protein, S100ß, and the excitatory amino acid transporter 1 and secreted neurotrophic factors (NTF) such as brain-derived neurotrophic factor, vascular endothelial growth factor, glial cell line-derived neurotrophic factor, and insulin-like growth factor 1. Conditioned medium of the induced cells rescued motor neurons from hypoxia or oxidative stress in vitro, suggesting a neuroprotective effect mediated by soluble factors. Given the neuronal support (NS) ability of the cells, the differentiated cells were termed hOMSC-NS. Rats subjected to sciatic nerve injury and transplanted with hOMSC-NS showed improved motor function after transplantation. At the graft site we found the transplanted cells, increased levels of NTF, and a significant preservation of functional neuromuscular junctions, as evidenced by colocalization of α-bungarotoxin and synaptophysin. Our findings show for the first time that hOMSC-NS generated from oral mucosa exhibit neuroprotective effects in vitro and in vivo and point to their future therapeutic use in neural disorders.

Stem cells of the lamina propria of human oral mucosa and gingiva develop into mineralized tissues in vivo.

AIMS: To characterize the mineralized tissue formed constitutively in the supracalvarial region of scid mice by a primitive stem cell population (hOMSC) derived from the lamina propria of the human oral mucosa and gingiva. MATERIAL AND METHODS: Fibrin-hOMSC constructs were cultured for 14 days at which time point they were analysed for the expression of osteoblastic/cementoblastic markers and implanted between the skin and calvaria bones into scid mice. After 8 weeks, the animals were sacrificed and the implantation sites analysed. RESULTS: Two-week-old cultures of fibrin-hOMSC constructs expressed osteogenic/cementogenic markers at the gene level. Macroscopic and radiographic examinations revealed mineralized masses at the implantation sites of fibrin-hOMSC constructs. Histology, histochemistry and immunofluorescence showed mineralized masses consisting of avascular cellular and acellular matrices that stained positively for collagen, Ca, cementum attachment protein, cementum protein 1, bone sialoprotein, alkaline phosphatase, osteocalcin, amelogenin and ameloblastin. Positive anti-human nuclear antigen indicated the human origin of the cells. Atomic force microscopy depicted long prismatic structures organized in lamellar aggregates. CONCLUSIONS: Within the limitation of this study, the results indicate for the first time that fibrin-hOMSC constructs are endowed with the constitutive capacity to develop into mineralized tissues that exhibit certain similarities to cementum and bone.

Randomized controlled trial on lateral augmentation using two collagen membranes: morphometric results on mineralized tissue compound.

BACKGROUND: Guided bone regeneration is considered an effective tool for gaining mineralized tissue either at exposed implant surface or in deficient alveolar ridge areas before implant placement. MATERIAL AND METHODS: Customized casts obtained following impression taking at surgery and re-entry allowed for morphometric assessment of alveolar ridge alterations 6 months after one-stage augmentation of bone dehiscences. In a randomized pilot study using biphasic calcium phosphate tests (n=17) received treatment with ribose cross-linked collagen membranes (RCLM), whereas controls (n=20) received non-cross-linked membranes. The primary endpoint was to quantify the effect of membrane type on dimensional changes in bone margins at crestal level of endosseous implants. RESULTS: Soft tissue dehiscencies occurred at 70.5% and 55% frequency for tests and controls, respectively. Gain in clinically hard newly mineralized tissue at the crestal level was significantly higher in test group in lateral (1.8 versus 0.7 mm; p=.046) and in vertical dimensions (1.1 versus 0.2 mm; p=.035) compared with controls. Second measurement obtained at the border of reflected flap revealed no significant difference between groups (3.0 versus 2.1 mm; p=0.57) for lateral dimension. CONCLUSIONS: Both collagen devices were effective in bone augmentation. RCLMs supported mineralization process and remodelling even in sites showing compromised healing as indicated by morphometric outcome.

The lamina propria of adult human oral mucosa harbors a novel stem cell population.

The highly regenerative capacity of the human adult oral mucosa suggests the existence of a robust stem cell (SC) population in its lamina propria (OMLP). The purpose of this study was to characterize the availability, growth, immunophenotype, and potency of this presumable SC population. Cells positive for the embryonic stem cell transcription factors Oct4 and Sox2 and for p75 formed distinct cord-like structure in the OMLP. Regardless of donor age, trillions of cells, termed human oral mucosa stem cells (hOMSC), 95% of which express mesenchymal stromal cell markers, were simply, and reproducibly produced from a biopsy of 3-4 x 2 x 1 mm(3). A total of 40-60% of these cells was positive for Oct4, Sox2, and Nanog and 60-80% expressed constitutively neural and neural crest SC markers. hOMSC differentiated in culture into mesodermal (osteoblastic, chondroblastic, and adipocytic), definitive endoderm and ectodermal (neuronal) lineages. Unexpectedly, hOMSC treated with dexamethasone formed tumors consisting of two germ layer-derived tissues when transplanted in severe combined immune deficiency mice. The tumors consisted of tissues produced by neural crest cells during embryogenesis-cartilage, bone, fat, striated muscle, and neural tissue. These results show that the adult OMLP harbors a primitive SC population with a distinct primitive neural-crest like phenotype and identifies the in vivo localization of putative ancestors for this population. This is the first report on ectodermal- and mesodermal-derived mixed tumors formation by a SC population derived from a nonmalignant somatic adult human tissue.

Long-term efficacy of a novel ribose-cross-linked collagen dermal filler: a histologic and histomorphometric study in an animal model.

BACKGROUND: Degradation and loss of the three-dimensional shape are the major causes of limited functional longevity of dermal fillers made of natural polymers as collagen and hyaluronic acid. OBJECTIVE: This study assessed the functional longevity of a new ribose-cross-linked collagen filler during 24 months in an animal model. METHODS: Ribose-cross-linked collagen (Evolence, Colbar Life Sciences Ltd), glutaraldehyde-cross-linked collagen (Zyplast, Inamed Inc.), and non-cross-linked collagen (Zyderm, Inamed Inc.) were injected in the rabbit ear dermis. Biopsies obtained at 1, 6, 12, and 24 months were histomorphometrically assessed for shape preservation and cell repopulation. RESULT: The three-dimensional shape of Evolence remained stable during 24 months. Zyderm and Zyplast lost their three-dimensional shape after 6 months. Although the cell density in Evolence remained stable over time, that in Zyplast and Zyderm decreased significantly at 12 and 24 months. CONCLUSION: Ribose-cross-linked collagen is endowed with a higher functional longevity as assessed in an animal model when compared with the most used collagen-based dermal fillers.

A two-stage phase I trial of Evolence30 collagen for soft-tissue contour correction.

BACKGROUND: The ideal dermal filler should be nonpermanent but with a durable effect lasting between 1 and 2 years, which is not the case with the resorbable fillers that are currently available. Evolence30 is a new, porcine-derived collagen gel based on the Glymatrix cross-linking technology, which results in a more natural and longer-lasting collagen product. METHODS: In this first clinical trial of Evolence30 (30 mg/ml), the safety and efficacy of this new filler were tested and compared with those of Zyplast (bovine cross-linked) collagen, after treatment of nasolabial folds in 12 volunteers. Safety assessments included two hypersensitivity tests, physical examination of injections sites, punch biopsies for histopathology, adverse events, and blood sample analysis. The seven-grade, validated Modified Fitzpatrick Wrinkle Scale was used by three independent blinded assessors to evaluate efficacy. RESULTS: No treatment-related adverse events were reported. Only transient erythema was observed in both treated sides, and there were no abnormal laboratory findings. None of the sera contained immunoglobulin (Ig) M, IgA, or IgE antibodies against porcine collagen at any time during the study. Initially, Evolence30 and Zyplast improved wrinkle severity to a similar extent. However, in an average follow-up of 18 months, assessment by the blinded assessors showed that the treatment effect on the Evolence30-treated side was superior in 9 of the 11 participants who were treated (p = 0.022). CONCLUSIONS: Evolence30 is a new, porcine-derived collagen product based on the Glymatrix cross-linking technology that enables a safe and effective correction of the nasolabial folds. This correction lasts significantly longer than that with Zyplast.

Gene profile in periodontal ligament cells and clones with enamel matrix proteins derivative.

AIM: Evaluate enamel matrix proteins derivative effect on gene expression profiles in cultured human periodontal ligament cell population and its clones. MATERIAL AND METHODS: Human periodontal ligament (PDL) cells were explanted. Cell cloning was performed and clones classified into fibroblastic (FB) and mineralized tissue forming (MTF) according to their capacity to express alkaline phosphatase and form mineralized tissue. All cell cultures were grown for 7 days, with and without enamel proteins added to the medium. Following RNA extraction, expression profiling was performed by hybridization with a DNA micro-array. Selected genes differed from the control at a significant level smaller than p<0.01. RESULTS: Enamel proteins induced major qualitative changes in mRNA expression in all PDL cell populations, differently affecting the entire PDL cell population and its clones. In the entire PDL cell population, enamel proteins significantly enhanced PDL cell function, with a general effect on enhanced cell functional metabolism. CONCLUSIONS: Enamel proteins enhanced gene expression responsible for protein and mineralized tissue synthesis in the entire PDL population. In the MTF clones, nucleic acid metabolism, protein metabolism and signal transduction related genes were up-regulated, while in the FB clones, up-regulated genes were related to cell adhesion, nucleic acid metabolism and signal transduction.

Healing of dehiscence-type defects in implants placed together with different barrier membranes: a comparative clinical study.

OBJECTIVE: Premature exposure of membranes used in guided bone regeneration (GBR) results in decreased bone formation. The effect of an expanded polytetrafluoroethylene (e-PTFE) and two collagen membrane on bone healing of buccal dehiscence defects around implants in cases with and without premature membrane exposure was clinically evaluated. METHODS: Three groups were established: Group OS (Ossix, n=73 implants, 41 patients), Group BG (Bio-Gide, n=53 implants, 28 patients) and Group GT (e-PTFE, Gore-Tex, n=34 implants, 17 patients). Defect height and width were measured at the time of implant placement and at second stage surgery. Surface area was calculated as half ellipses. When several implants were placed simultaneously, a mean of their defect width and height was calculated. RESULTS: Mean percentage reduction of defect area (92.2+/-13.78% Group OS, 94.6+/-6.69% Group BG, and 97.3+/-4.91% Group GT) and height (81.6+/-23.19%, 85.4+/-12.26%, and 93.4+/-9.39% respectively) did not show statistically significant differences between groups. Differences between groups were not statistically significant for all parameters when cases without spontaneous membrane exposure were compared. However, differences were significant when spontaneous membrane exposure occurred. Mean percentage reduction of defect area among cases where membrane exposure occurred was 91.5+/-10.86% Group OS, 71.5+/-8.61% Group BG, and 73.7+/-13.97% Group GT. Mean percentage reduction of defect height among cases with membrane exposure was 76.4+/-18.28%, 53.4+/-9.86%, and 49.4+/-11.05%, respectively. CONCLUSIONS: Premature exposure of membranes and subsequent and consequent exposure of implants results in impaired bone healing. Certain barrier membranes, as used in group OS, are apparently capable of supporting gingival healing even when prematurely exposed that could be advantageous in GBR procedures.

Anti-cementoblastoma-derived protein antibody partially inhibits mineralization on a cementoblastic cell line.

The effect of human anti-cementoblastoma-derived protein antibody during cementogenesis in vitro was investigated by using human cementoblastoma-derived cells. Cultures treated with 5 microg/ml of CP antibody from day 1 to day 15 revealed a significant decrease in alkaline phosphatase activity (ALP) 40% (p < 0.005), 44% (p < 0.001), 49% (p < 0.1), and 45% (p < 0.02) at 9, 11, 13, and 15 days, respectively. Immunoexpression of osteopontin revealed that in cultures treated with anti-CP antibody, the positive number of cementoblastoma cells was reduced by 87, 83, 69, and 52% at 5, 7, 9, and 11 days, respectively. Bone sialoprotein immunoexpression showed a decrease in positive cells of 82, 51, 60, 80, 83, and 87% at 5, 7, 9, 11, 13, and 15 days, respectively, as compared to controls. The Ca/P ratio of the mineral-like tissue deposited in vitro by cementoblastoma cells revealed that control cultures had a Ca/P ratio of 1.45 and 1.61 at 5 and 15 days, whereas experimental cultures revealed a Ca/P ratio of 0.50 and 0.79 at 5 and 15 days, respectively. Electron diffraction patterns showed inner double rings representing D-spacing that were consistent with those of hydroxyapatite in both control and experimental cultures. Examination of the crystallinity with high resolution transmission electron microscopy showed homogeneous and preferential spatial arrangement of hydroxyapatite crystallites in control and experimental cultures at 15 days. Atomic force microscopy images of control cultures at 5 and 15 days revealed small granular particles and grain agglomeration that favored the formation of crystalline plaques with a lamellar-like pattern of the mineral-like tissue. Experimental cultures at 5 and 15 days showed tiny and homogeneous granular morphology. The agglomerates maintained spherical morphology without organization of needle-like crystals to form plaque-like structures. Based on these findings, it is hypothesized that cementoblastoma-derived protein may be associated to crystal growth, compositional and morphological features during the mineralization process of cementum in vitro.

The effect of bacterial endotoxin on the early tensile strength of healing surgical wounds.

Wound healing in the oral cavity occurs in a bacteria-rich environment, which may affect its outcome. Furthermore, it takes place where forces are frequently applied to the healing tissue. The effect of bacterial endotoxin on the development of tensile strength in healing wounds was studied using surgical skin wounds in rats as a model. Collagen membranes soaked with 0.01 microg of bacterial endotoxin were inserted into surgical skin wounds, and their effect was studied on days 6 and 10. Membranes with no endotoxin served as controls. Endotoxin inhibited the early development of tensile strength in 6 days, healing wounds by 38%, whereas the collagen membrane alone had no effect. Dexamethasone (0.5 mg/kg every 72 h) had a suppressive effect on the development of tensile strength in healing noncontaminated wounds, but not in those containing bacterial endotoxin. These results suggest that bacterial endotoxin may interfere with the early healing of wounds. Understanding the mechanisms of this inhibition may result in treatments that will allow this response to be faster and more reproducible.

Establishment of a rat long-term culture expressing the osteogenic phenotype: dependence on dexamethasone and FGF-2.

Rat stromal bone-marrow cells cultured in the presence of dexamethasone, ascorbic acid, beta-glycerophosphate, and fibroblast growth factor-2 (FGF-2) express the osteogenic phenotype (Pitaru et al., J. Bone Miner. Res. 8:919-929, 1993). The purpose of this study was to establish a long-term homogeneous culture expressing the osteogenic phenotype. The cultures were routinely passaged every 5 days in the absence or presence of either or both dexamethasone and FGF-2, and the cumulative doubling number and the expression of the osteogenic phenotype were determined. Cultures treated with dexamethasone (10(-7) M) ceased proliferation and only upon addition of FGF-2 (3 ng/ml) was a spontaneous immortalization achieved, as expressed by sustained proliferation for about 1 year, with a doubling time of 22 h and more than 300 doublings in 72 passages. Both FGF-2 and dexamethasone are required and act synergistically to maintain cell propagation, alkaline phosphatase expression, and osteocalcin secretion; however, protein content was FGF-2 dependent and the mineralization was dexamethasone dependent. Repetitive single-cell cloning tested the homogeneity and stability of the cells expressing the osteogenic phenotype in these long-term cultures. It was shown that 25% to 50% of subclones derived from clones with an osteogenic phenotype do not further express the osteogenic phenotype. In conclusion, we have established a spontaneously immortalized dexamethasone- and FGF-2-dependent rat stromal bone-marrow-derived long-term culture expressing the osteogenic phenotype. The cultures tend to lose the osteogenic phenotype, and dexamethasone supports the long-term preservation of the osteogenic phenotype.

Histological assessment of augmented jaw bone utilizing a new collagen barrier membrane compared to a standard barrier membrane to protect a granular bone substitute material.

Successful bone augmentation requires predictable space maintenance and adequate exclusion of those cells that lack osteogenetic potential from the defect area. Natural bone mineral is considered to be osteoconductive and is used as space maker in combination with membrane barrier techniques. The aim of this study was to compare qualitative histological results achieved by using deproteinized bovine bone mineral (DBBM) as a space maintainer and a new collagen barrier (Ossix, test group) vs. the same bone substitute and the standard e-PTFE membrane (Gore-Tex), control group). Twenty-eight patients were randomly assigned to the test or the control group. Seven months after augmentation procedures, biopsies were obtained at reentry and were analysed histomorphometrically. In all, 14 specimens of group I (test group, Ossix) and 13 specimens of group II (controls, PTFE-membranes) showed close qualitative similarity of their histologies. Histomorphometrically, total mineralized bone area was 42% +/- 18% in group I vs. 39% +/- 15% in group II. The unmineralized tissue area was 44% +/- 15% vs. 46% +/- 12% and the area of DBBM remnants 14% +/- 9% and 15% +/- 12%, respectively. The differences were statistically nonsignificant (Mann-Whitney test). The occurrence of barrier exposure did not interfere with the histological outcome either in the test or in the control group. The new collagen barrier combined with the DBBM provided qualitative bone regeneration comparable to the standard e-PTFE material combined with the same mineral.